Colony stimulating factor-1 (CSF-1) is absolutely required for normal osteoclastogenesis. Alternative splicing of the CSF-1 gene results in production of soluble (sCSF-1) and cell-surface (mCSF-1) isoforms. While both in vivo and in vitro studies have established that sCSF-1 is bioactive in bone, our data were among the first to demonstrate skeletal activity for mCSF-1. We have found that selective inhibition of mCSF-1 in osteoblasts markedly suppresses osteoblast-mediated osteoclastogenesis. Selective expression of a mCSF-1 transgene in osteoblasts leads to osteopenia in normal mice and completely rescues the osteopetrotic phenotype of the op/op mouse. While these data unequivocally establish that both isoforms are active in bone, it remains unclear if they subserve distinct functions. Further, whether CSF-1 is simply a survival factor for osteoclast precursors, or whether it has pro-differentiation effects remains controversial. To address these issues we will pursue the following Specific Aims: (1) undertake isoform-specific targeted deletion of the two CSF-1 isoforms in osteoblasts and examine the impact of this intervention on bone; (2) explore the hypothesis that estrogen-deficiency bone loss is, in part, mediated by selective up-regulation of mCSF-1 by examining the effect of estrogen withdrawal in animals with conditional deletion of mCSF-1 in osteoblasts. We will also begin to explore the molecular mechanism by which estrogen withdrawal selectively up-regulates mCSF-1; (3) continue our studies in transgenic mice selectively expressing sCSF-1 or mCSF-1 in osteoblasts, by examining the effects of PTH-infusion and ovariectomy in these animals. We hypothesize that increased expression of mCSF-1 in bone will sensitize the skeleton to the resorbing actions of PTH and estrogen withdrawal, particularly given that estrogen withdrawal selectively up-regulates the mCSF-1 isoform; and (4) since activation of the transcription factor mitf by CSF-1 is required for full osteoclast maturation, gene profiling will be undertaken in osteoclast precursors from wild-type and mi/mi mutant mice, both before and after treatment with CSF-1, as a way of identifying CSF-1-dependent pathways required for osteoclastogenesis. These studies will (1) improve our understanding of CSF-1's role in physiologic and pathologic bone resorption, and (2) identify critical CSF-1-dependent steps in osteoclastogenesis which may provide novel targets for drug discovery.